亚硒酸钠对猪卵母细胞体外成熟及其胚胎发育潜能的影响

doi:10.3864/j.issn.0578-1752.2024.17.013

Abstract

Abstract:

【Objective】The aim of this study was to investigate the effects of sodium selenite (SS) on the in vitro maturation of porcine oocytes and their embryonic developmental potential, and to conduct a preliminary analysis of its mechanism, so as to provide the theoretical references for the improvement of the in vitro maturation system of porcine oocytes.【Method】After culturing porcine cumulus-oocyte complexes (COCs) in maturation medium containing different concentrations of SS (0, 20, 40, 60, and 80 nmol·L^-1) for 44 hours, observations were made, and cumulus cell expansion and oocyte maturation status were collected. Cumulus cell RNA was extracted, and oocytes were subjected to parthenogenetic activation (PA) treatment. The cleavage rate and blastocyst rate of parthenogenetic embryos were statistically analyzed at 48 and 168 hours of in vitro culture. Real-time fluorescence quantitative PCR and observation were used to detect cumulus cell expansion index (CEI), expression of cumulus expansion-related genes (Has2, Ptgs2), first polar body (PB1) extrusion rate, and cleavage rate and blastocyst rate of parthenogenetic embryos, to investigate the effects of different concentrations of SS on oocyte maturation, early embryo development, and cumulus cell expansion, and to determine the optimal concentration of SS. Total antioxidant capacity of COCs, glutathione (GSH) content, malondialdehyde (MDA) levels, and expression of antioxidant-related genes (CAT, PRDX2) in oocytes were detected by spectrophotometry and RT-qPCR to explore the effect of adding SS to the maturation medium on oocyte antioxidant capacity. Immunofluorescence technology was combined to detect the total number of cells in blastocysts and the expression of pluripotency genes (Nanog, Sox2) in blastocysts, to investigate the effect of SS on the developmental potential of parthenogenetic embryos.【Result】The RT-qPCR results showed that the addition of different concentrations of SS significantly promoted the expression of the PTGS2 gene (P<0.05), and at a concentration of 40 nmol·L^-1, the expression of the HAS2 gene, CEI, PB1 extrusion rate of oocytes, and blastocyst rate of early embryos were all significantly promoted (P<0.05). SS at concentrations of 40, 60, and 80 nmol·L^-1 promoted oocyte cleavage, with significant promotion observed at a concentration of 40 nmol·L^-1 (P<0.05). Based on those results, the optimal concentration of SS adding to the in vitro maturation medium for porcine oocytes was determined to be 40 nmol·L^-1. The results of antioxidant capacity testing showed that SS significantly increased the total antioxidant capacity of COCs, the level of GSH in oocytes, and the expression of antioxidant genes CAT and PRDX2, while significantly reducing MDA content (P<0.05). Immunofluorescence and PCR results showed that the number of inner cell mass in blastocysts and the expression of Nanog were increased in the SS group (P<0.05).【Conclusion】The addition of SS to the in vitro maturation medium of oocytes promoted cumulus cell expansion, enhanced oocyte maturation rates, and improved the developmental potential of matured oocytes. The beneficial effects of SS on the in vitro maturation of oocytes might be associated with its antioxidant properties.

Key words: sodium selenite (SS), porcine oocytes, in vitro maturation (IVM), oxidative stress, parthenogenetic activation

ZHANG Bing, YANG YanYan, FENG QianHui, SHI Wen, FANG YiZhen, HUANG JiaBao, SHI DeShun. Effects of Sodium Selenite on the in vitro Maturation of Porcine Oocytes and Their Embryonic Development Potentials[J].Scientia Agricultura Sinica, 2024, 57(17): 3482-3493.

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基因 Gene	引物序列 Primer sequence	长度 Length (bp)	GenBank登录号 Accession No.
GAPDH	F: TATGATTCCACCCACGGCAAG R: CCACAACATACGTAGCACCAG	145	NM_001206359.1
PTGS2	F: CAGGCTGATACTGATAGGAGAAA R: CTGGTTGAAAAGCAGCTCTGG	112	NM_214321.1
HAS2	F: ATGACAGGCATCTAACGAAC R: TTGTACAGCCACTCTCGGAA	157	NM_214053.1
CAT	F: GCTGAGTCCGAAGTCGTCTA R: GTCAGGATATCAGGTTTCTGCG	173	NM_214301.2
PRDX2	F: TTCGGGCCGAGCATAAAAGG R: AGGCCATGACTGAAAGCTGC	119	NM_001244474.1
NANOG	F: TCCACAAGCCCCAGAGTAAA R: GCCTCTGAAATCTGTCGTTG	145	NM_001129971.2
SOX2	F: AAGAACAGCCCAGACCGAGT R: CCGTCTCCGACAAAAGTTTCCAC	154	NM_001123197.1

浓度 Concentration (nmol·L^-1)	卵母细胞数 No. of oocytes	卵裂数 No. of cleavage	卵裂率 Rate of cleavage	囊胚数 No. of blastocyst	囊胚率 Rate of cleavage
0	369	295	79.47±1.44c	73	24.74±1.88b
20	362	307	82.03±1.94bc	86	28.22±1.34ab
40	406	365	87.7±1.59a	114	31.49±1.19a
60	372	320	84.49±1.00b	89	28.23±0.59ab
80	376	323	84.41±1.59b	82	26.27±2.81b

Effects of Sodium Selenite on the in vitro Maturation of Porcine Oocytes and Their Embryonic Development Potentials

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